The oxygen carrying portion of red blood cells is the protein hemoglobin. Hemoglobin is a tetrameric protein molecule composed of two identical alpha globin subunits (.alpha..sub.1, .alpha..sub.2), two identical beta globin subunits (.beta..sub.1, .beta..sub.2) and four heme molecules. A heme molecule is incorporated into each of the alpha and beta globins to give alpha and beta subunits. Heme is a large macrocyclic organic molecule containing an iron atom; each heme can combine reversibly with one ligand molecule such as oxygen. In a hemoglobin tetramer, each alpha subunit is associated with a beta subunit to form two stable alpha/beta dimers, which in turn associate to form the tetramer. The subunits are noncovalently associated through Van der Waals forces, hydrogen bonds and salt bridges.
Hemoglobin in solution can be used, for example, as a blood substitute, as a therapeutic for enhancing hematopoiesis, as a means of delivering oxygen or enhancing oxygen delivery to tissues, for hemoaugmentation, for the binding or delivery of nitric oxide or other non-oxygen ligands, as a drug delivery vehicle, as a cell culture additive, as a reference standard, and as an imaging agent. However, storage of hemoglobin solutions can be problematic. Proteins in solution can form aggregates upon long term storage, changes in temperature during storage, or mechanical agitation (Cleland, et al., Crit. Rev. Ther. Drug Carrier Systems 10: 307-377 (1993). To address these problems, many unique formulations have been developed for the stabilization of different proteins in solution. For example, both naturally derived and recombinantly produced proteins have been formulated in solutions containing disaccharides and amino acids (factor VII or factor IX solutions described in PCT Publication WO 91/10439 to Octapharma), human serum albumin (interleukin-2 solutions described in U.S. Pat. No. 4,645,830 to Yasushi et al.), and glycine, mannitol and non-ionic surfactants (human growth hormone solutions described in U.S. Pat. No. 5,096,885 to Pearlman et al.). Although some general guidance is available for the determination of suitable components for formulations for protein solutions, because of the unique nature of individual proteins, no single formulation is suitable for all different proteins. Indeed, Cleland et al. (1993) state that the creation of a formulation that minimizes protein degradation is difficult because there are many factors that interact to determine protein degradation in a formulation. They go on to state that "protein degradation . . . cannot be predicted a priori and must be determined for each protein".
To extend the storage stability of hemoglobin solutions by limiting autooxidation, hemoglobin has been formulated with reducing agents such as cysteine or dithionite, mannitol, glucose and/or alpha tocopherol (Shorr et al., PCT Publication WO 94/01452), in saline solutions or lactated Ringer's solutions that have been modified by the addition of, for example ascorbate, ATP, glutathione and adenosine (Feola et al., PCT Publication WO 91/09615; Nelson et al, PCT Publication WO 92/03153), under deoxygenated conditions with no exogenous reductants (Kandler and Spicussa, PCT Publication WO 92/02239), or in the presence of reducing enzyme systems (Sehgal et al., U.S. Pat. No. 5,194,590). These hemoglobin formulations have been designed to minimize autooxidation of the protein molecule, but none have been designed that specifically reduce the aggregation of the hemoglobin molecules during storage.
Nonetheless, the aggregation of hemoglobin molecules during storage poses significant problems. Moore et al., Art. Org. 16: 513-518 (1992) caution that hemoglobin should not be stored in the frozen state due to the formation of aggregates or precipitates. Moreover, the formation of aggregates in hemoglobin solutions agitated at room temperature has been observed in numerous formulations (Pristoupil and Marik, Biomat. Art. Cells. Art. Org., 18: 183-188, 1990; Adachi and Asakura, J. Biol. Chem., 256: 1824-1830, 1981; Adachi and Asakura, Biochem. 13: 4976-4982,1974). This aggregation of the hemoglobin protein molecule typically does not occur as a result of autooxidation of the hemoglobin heme iron, but rather by interaction of the hemoglobin molecules (Adachi et al., Fed. Proc. 35: 1392 (1976). Aggregates in hemoglobin solutions can increase immunogenicity, reduce functionality and reduce the activity of the protein solution (Cleland et al., supra; Feola et al., Biomat. Art. Cells Art. Org. 16: 217-226 (1988).
Accordingly, there is a need for hemoglobin compositions stabilized against the formation of aggregates. The present invention satisfies this need and provides related advantages as well.